The major objective of this research is to learn the detailed mechanism by which muscle pyruvate kinase polarizes the carbonyl group of pyruvate facilitating its enolization, and catalyzes the transfer of the Gamma-phosphoryl group of ATP to the enolate of pyruvate. We also wish to learn how the rates of these processes are controlled by fructose diphosphate and by phosphorylation of the enzyme in the allosteric hepatic pyruvate kinase. Also under investigation are the mechanistically related phosphoryl transfer reactions catalyzed by adenylate kinase and creatine kinase and the enolization reaction catalyzed by glyoxalase I. Nuclear magnetic resonance methods using paramagnetic probes, nuclear Overhauser effects, and computerized conformational search procedures are used to elucidate the roles of the essential metal cofactors, the conformations, arrangements, and exchange rates of enzyme-bound substrates, and the nature of the functional groups on the enzyme(s) which interact with the substrates. Steady state kinetics, chemical modification of enzymes and X-ray absorption (EXAFS) methods supplement the magnetic resonance studies and provide additional structural and mechanistic information. In the case of adenylate kinase, we are examining in great detail, the interaction of the substrate ATP with a 44 amino acid peptide fragment of this enzyme (1-44) which binds ATP with essentially the same affinity as that of the entire 193 amino acid enzyme. We are also investigating the interaction of the other substrate AMP with a smaller peptide fragment (171-193). The peptide and nucleotide conformations and their interactions in these fragments will be compared with those in the intact enzyme to determine whether "isolated active sites" are in hand. On glyoxalase I we are studying the enolization mechanism and the conformations of the enzyme-bound tripeptide coenzyme, glutathione and its derivatives. The purpose of studying several enzymes which catalyze chemical reactions of the same class is to elucidate general principles of enzyme chemistry, and to develop and critically compare various spectroscopic approaches to enzyme structure and mechanism.